Hyaluronic acid or hyaluronan (HA) is a large negatively charged glucosaminoglycan consisting of repeating disaccharides of N-acetylglucosamine and -glucuronic acid. This polymer is ubiquitous in the extracellular matrix and is the major component of skin, cartilage and brain tissue. In addition to its known macrostructural functions, it also performs physicochemical functions, for example, by acting as a lubricant in the synovial fluid in joints.
Synthesis of HA has been associated with the morphogenesis of many tissues, with wound repair, tumour invasion and cellular immune function (Toole, B. P., Connect. Tissue Res. 10: 93-100, 1982; Pauli, B. V. et al., Cancer Met. Rev. 2: 129-152, 1983; Toole, B. P. et al., Ciba Found. Symp. 143: 150-169, 1989; Turley, E. A., Cancer Met. Rev. 3: 325-339, 1984; Iozzo, R. V., Lab. Invest. 53: 373-396, 1985; Weigel, P. H. et al., J. Theol. Biol. 119: 219-234, 1986; Weigel, P. H. et al., Ciba Found. Symp. 143 248-264, 1989; Boudreax, N. et al., Dev. Biol. 143: 235-247, 1991; Turley, E. A., Cancer Met. Rev. 11: 21-30, 1992). The underlying mechanism of action at the cellular level is believed to involve the ability of HA to elicit receptor-mediated alterations of cell motility. High affinity HA receptors have been identified and characterized on a variety of cell types and these are namely the receptor for hyaluronan mediated mobility (or RHAMM) (Turley, E. A. et al., J. Cell. Biol. 112: 1041-1047, 1991; Hardwick, C. et al., J. Cell. Biol. 117: 1343-1350, 1992; Yang, B. et al., J. Biol. Chem. 268: 8617-8623, 1993), intercellular adhesion molecule-1 (or ICAM-1) (McCourt, P. A. G. et al., J. Biol. Chem. 269: 30081-30084, 1994) and CD44 (Underhill, C. B. et al., J. Biol. Chem. 262: 13142-13146, 1987; Stamenkovic, I. et al., Cell 56: 1057-1062, 1989; Aruffo, A. et al., Cell 61: 1303-1313, 1990; Lesley, J. et al., Exp. Cell Res. 187: 224-233, 1990; Miyake, K. et al., J. Exp. Med. 172: 69-76, 1990). Both RHAMM and CD44 have been shown to be associated with cell locomotion, cell proliferation and differentiation. Other HA-binding proteins in the extracellular milieu have also been identified and they are namely link protein, aggrecan, versican, GHAP, collagen type VI and TSG-6.
In tissue trauma, an acute, transient increase in production of HA is observed which is accompanied by an increase in expression of HA receptors such as the receptor for hyaluronan mediated mobility (RHAMM) and CD44. The in vivo physiological implications of these molecular events has not been fully elucidated although an increased production of HA and its accumulation has been shown to regulate locomotion of fibroblasts, inflammatory cells and epidermal cells depending upon the concentration of HA used. Further, HA affects collagen fibril formation and white blood cell phagocytic function, peroxide generation and cytokine expression. Prior research studies have demonstrated that high concentrations of HA act as a RHAMM antagonist to injured tissues including skin burns, ulcers, ruptured tympanic membranes and abraded cornea by reducing tissue fibrosis (Goz, K. L. and Benfield, P., Drugs 47: 536, 1994; King, S. R. et al., Surgery 109: 76, 1991; Riquelme-Saquier, J. L., Dev. Ophthamol. 22: 50, 1991; Chung, J. H. et al., Exp. Eye Res. 48: 569, 1989; Hellstrom, S. and Laurent, C., Acta Otolaryngol. 442 (Suppl.): 54, 1987; Retanda, G. G., Ital. Dermatol. Venereol. 120: 71-75, 1985; Abatangelo, G. et al., J. Surg. Res. 35: 410, 1983). Fibrosis of adult tissues after trauma is a serious clinical problem that can result in pathologies including malfunction of tissues due to keloids, hypertrophic scars, anatomonic strictures, intra-abdominal adhesions, cirrhosis of the liver, neurological deficits following spinal cord injury, valvular heart diseases, burn-injured joints as well as failure of anastomosis and adhesions following surgery (Bleacher, J. C. et al., Dermatologic Clinics 11: 677-683, 1993; Clark, R. A. et al., Am. J. Med. Sci. 306: 42-48, 1993; Hebda, P. A. et al., Dermatologic Clinics 11: 685-696, 1993; Adzick, N. S. and Longaker, M. T., Ann. Surg. 215: 3-7, 1992; Folkman, J., Ann. Surg. 215: 1-2, 1992).
Antagonism of RHAMM by antibodies and peptides has also been demonstrated to inhibit HA-promoted cell locomotion (International application PCT/CA93/00158 published as WO 93/21312). For instance, HA-promoted fibroblast locomotion was inhibited by application of a polyclonal or monoclonal antibody against the HA-receptor complex (HARC).
Tumorigenesis is commonly manifested by an uncontrolled proliferation of cells, and the metastatic spread of tumour tissue is associated with the ability of these cells to locomote and invade. Oncogenes and tumour suppressor genes are important factors in the control of tumour cell growth, but extracellular matrix (ECM) molecules such as HA and their receptors also play significant roles. HA and RHAMM have been shown to regulate cell proliferation and differentiation and are implicated in cell transformation and tumour metastasis.
The transforming oncogene H-ras promotes cell locomotion by enhancing the formation and release of autocrine motility factors, growth factors and extracellular matrix (ECM) molecules such as HA (Toole, B. P., Curr. Opin. Cell Biol. 2: 839-844, 1990; Stoker, M. et al., Biochim. Biophys. Acta 1072: 81-102, 1991; Hardingham, T. E. and Fosang, A. J., FASEB J. 6: 861-870, 1992; Laurent, T. C. and Fraser, J. R. E., FASEB J. 6: 2397-2404, 1992; Pilarski, L. M. et al., Leuk, Lymp. 14: 363-374, 1994). Enhancement of ras-transformed cell locomotion by HA has been found to depend on the presence of a HA-receptor complex termed HARC occurring at the cell surface or released as soluble proteins (Hall, C. L. et al., Cell 82: 1-20, 1995; Hall, C. L. and Turley, E. A., J. Neuro-Oncol. 26: 221-229, 1995; Turley, E. A. et al., Blood 81: 446-453, 1993; Turley, E. A. et al., Exp. Cell Res. 207: 277-282, 1993; Turley, E. A., Cancer Metast. Rev. 11: 21-30, 1992; Turley, E. A. et al., J. Cell Biol. 112: 1041-1047, 1991). Turley et al. (Exp. Cell Res. 207: 277-282, 1993) reported that such HA-promoted cell locomotion was inhibited by monoclonal antibodies specific to RHAMM thereby implicating RHAMM as the major HA-binding component of HARC in tumorigenesis and metastasis.
Under normal physiological conditions, RHAMM is not detectable on B-lymphocytes found in the blood, spleen or lymph node. Among B-cell malignancies, RHAMM is overexpressed on most terminally differentiated B-cells from multiple myeloma bone marrows, certain non-Hodgkin's lymphomas, and splenic hairy leukemic cells (Turley, E. A. et al., Blood 81: 446-453, 1993; Masellis-Smith, A. et al., Blood 87: 1891-1899, 1996). RHAMM is also overexpressed in breast carcinoma cells (Turley, E. A. et al., Exp. Cell Res. 207: 277-282, 1993; Hall, C. L. & Turley, E. A., J. Neuro-oncol. 26: 221-229, 1995), and in combination with an increased level of HA, are responsible for their enhanced motility and metastasis. Administration of RHAMM-transfected cells into animals results in spontaneous metastasis and formation of lung tumour colonies.
RHAMM was one of the first HA receptors to be isolated and characterized at the biochemical and molecular levels. It is an N-linked glycoprotein that binds HA with high affinity (Kd: 1 nM) and specificity. Several isoforms of RHAMM with different subcellular distribution have been identified. Isoforms found intracellularly and on the plasma membrane are designated iRHAMM and pRHAMM, respectively, and the secreted isoform is designated sRHAMM. The molecular structure of the various RHAMM isoforms may be differentially regulated by phosphorylation and/or glycosylation statuses. The precise roles of the RHAMM isoforms have not been fully elucidated, but it is believed that pRHAMM and sRHAMM elicit opposite activities and the net functional behaviour of a HA-RHAMM interaction depends at least in part on the balance of pRHAMM versus sRHAMM expressed by the cells involved.
Yang, B. et al. (J. Biol. Chem. 268: 8617-8623, 1993) have identified two discrete HA binding domains in RHAMM that occur at the carboxyl terminus of the protein. These domains are the only HA binding regions in the receptor protein and they each contribute approximately equally to the HA binding ability of RHAMM. Mutation studies have revealed that Domain I contains two sets of two basic amino acid residues spaced seven amino acids apart are important for HA binding of the receptor. Similarly, Domain II contains a lysine residue at position 423 and arginine at position 431 also spaced seven amino acids apart which are critical for HA binding activity. Collectively, these data predicted a generic binding motif with a structure of B.sup.1 -A.sub.n -B.sup.2 representing a minimal binding requirement for HA and RHAMM. B.sup.1 and B.sup.2 are the same or different basic amino acid residues and A.sub.n is a peptide sequence containing seven or eight amino acid residues which are the same or different and are neutral or basic amino acids. This generic binding motif was also found to be present in CD44, link protein and all other HA binding proteins discovered to date.
A full-length murine RHAMM cDNA has been cloned successfully from a GT11 3T3 cDNA expression library (Hardwick, C. et al., J. Cell Biol. 117: 1343-1350, 1992). Immunoblot analyses of cell lysates using antibodies to peptides encoded in the cDNA reacted specifically reacted with RHAMM protein. Using a fragment of the clone DNA sequence, a mouse fibroblast genomic library was screened to clone the genomic RHAMM gene which spans at least 20 kb and comprises 14 exons ranging in size from 75 to 1099 bp (Entwistle, J. et al., Gene 163: 233-238, 1995).
Similarly, a human RHAMM cDNA clone was also isolated successfully by a combination of screening a human breast cDNA expression library with the murine RHAMM cDNA as well as 5' RACE and reverse transcription-polymerase chain reaction using messenger RNA from the human breast cell line MCF-10A (Wang, C. et al., Gene 174: 299-306, 1996). The full-length human RHAMM cDNA encodes for a 725 amino acid protein and shares a 85% homology with the murine transcripts, RHAMM v4. More importantly, the HA binding motif B.sup.1 -A.sub.n -B.sup.2 which is shown to be critical for the signalling capability of RHAMM is 100% conserved between human and mouse.
PCT published patent application no. WO 93/21312 to the present inventor describes short peptides of nine or ten amino acid residues which mimic the HA binding motif of RHAMM. These RHAMM peptides possess the ability to bind HA and share a common generic peptide sequence represented by B.sup.1 -A.sub.n -B.sup.2 as described above.
However, the published application discloses key restrictions associated with the sequence of these RHAMM peptides with respect to their HA binding affinity. For example, it is clear from the patent publication that if A.sub.n is a peptide sequence containing less than seven or greater than eight amino acid residues, HA binding affinity is lost. Acidic amino acids are incompatible with HA binding as substitution of neutral or basic amino acid residues by acidic amino acid residues also abolishes HA binding affinity. Moreover, the basic amino acid histidine at the carboxy-terminal end is not compatible with HA binding which is indicated by the fact that replacement of the carboxy-terminal lysine or arginine by histidine completely destroyed HA-binding ability of the RHAMM peptides. In particular, a peptide with the sequence KLRSQLVHHH was unable to bind to HA.
PCT published patent application no. WO 97/24111 to the present inventor also describes HA-binding peptides consisting of dextrorotatory, D-amino acids and their ability to bind naturally occuring hyaluronic acid in the body which prevents hyaluronic acid from stimulating its receptors. Correspondingly, through the inhibition of hyaluronic acid receptor activation, said D-forms of HA-binding peptides were speculated to be useful when combined with a second medicine or therapeutic agent such as a surfactant for the treatment of herpes infection, an anti-microbial agent for the treatment of mononucleosis, dimethyl sulphoxide for AIDS therapy, insulin for the treatment of diabetes and a calcium channel blocker for the treatment of hypertension.
The invention of WO 97/24111 is distinct from the peptides and uses of the present invention in two major respects. First, based on past empirical evidence, it is well known that many physiologically and therapeutically important peptides of similar sizes to the peptides of the present invention exhibit significant stereospecificity in their biological actions. In many instances, subsitution of L-amino acid(s) by D-amino acid(s) or vice versa resulted in peptides that produce physiological effects that are opposite to those originally observed. Such a seemingly straightforward change of one or more amino acid residue(s) by its enantiomeric counterpart(s) can therefore create peptides that have distinctly different therapeutic uses.
For example, vasopressin is a nonapeptide and substitution of L-amino acid residue(s) to D-amino acid residue(s) in the SK&F vasopressin analogs dramatically reversed their bioactivity as vasopressin agonists to vasopressin antagonists (Albrightson-Winslow, C. et al., J. Pharmacol. Exp. Ther. 256: 335-340, 1991; Brooks, D. P. et al., Eur. J. Pharmacol. 160: 159-152, 1989). Similarly, enantiomeric substitutions of L- and D-amino acids in substance P, an undecapeptide, also dramatically changed its agonistic and antagonistic activities as well as the peptide's specificity to stimulate different receptor subtypes and to elicit totally different physiological responses (Cross, M. et al., Eur. J. Pharmacol. 291: 291-300, 1995; Dutta, A. S. et al., J. Med. Chem. 29:1163-1171, 1986; Dutta, A. S. et al., J. Med. Chem. 29:1171-1178, 1986). To further illustrate this phenomen, substance P with an L-proline at position 9 induced a marked scratching response in the rat but does not produce any response in colon smooth muscle. Conversely, substitution of this single amino acid by a D-proline resulted in a peptide that elicited no scratching response but produced sigificant contraction of colon smooth muscle (Piercey, M. F. et al., Life Sci. 36: 777-780, 1985).
Similarly, Casteels, P. and Tempst, P. reported the stereospecific requirements of apidaecin-type antibacterial peptides in which the L-enantiomer of apidaecin is a lethal non-poreforming bacteriotoxin but the D-enantiomer is completely devoid of antibacterial activity (Casteels, P. and Tempst, P., Biochem. Biophys. Res. Commun. 199: 339-345, 1994). Oren, Z. et al. also reported the detrimental effects of D-amino acid substitution on the alpha-helical structure of diastereomeric dodecapeptides and their antibacterial effects (Oren, Z. et al., J. Biol. Chem. 272: 14643-14649, 1997).
Furthermore, peptidyl dipeptide hydrolase catalyzes the hydrolysis of the decapeptide angiotensin I to the physiologically active octapeptide angiotensin II. This enzyme has been shown to exhibit an absolute stereospecific requirement for L-amino acid residues at the C-terminus of the angiotensin I peptide in order to achieve activation (Oshima, G. and Nagasawa, K., J. Biochem. 86: 1719-1724, 1979; Oparil, S. et al., Circ. Res. 29: 682-690, 1971).
In view of the above, the biological activities of one enantiomer of a given peptide are quite distinct from those of another enantiomer of the same peptide. The speculation of certain activities for the D-forms of the HA-binding peptides in WO 97/24111 therefore cannot be used to predict the clinical utilities of the L-forms of said peptides. This is supported by the fact that the L-peptides of the present invention, when given alone, are useful in the prevention of skin tissue fibrosis which is clearly in contrast to the combinational therapeutic regimens and the therapeutic areas described in WO 97/24111.
Second, notwithstanding the terminology "HA-binding peptides" and that the actions of the D-peptides in WO 97/24111 depend on their ability to inhibit HA interaction with its receptors, the L-forms of the peptides of the present invention do not elicit its biological actions by inhibiting HA interaction with its receptors (RHAMM and CD44). This aspect of the invention is demonstrated in greater detail below.